Project Summary Invasive fungal infections cause unacceptable mortality rates in neutropenic patients. Since survival of neutropenic patients with invasive fungal infections is linearly related to the patients' granulocyte counts, exogenous replacement of phagocytes is of great therapeutic potential for these infections. Although neutrophil transfusions have shown promising results, they fell out of favor in the early 1980s due to several technical difficulties. To address invasive fungal infections in neutropenic patients, we are developing a cell-based immunotherapy as an alternative to neutrophil transfusions. Our strategy is based on a human phagocytic cell-line, HL-60, which can be grown perpetually in vitro, but can then be activated to differentiate towards a mature neutrophil phenotype prior to infusion into an infected, myeloablated host. Activation of HL-60 cells into Activated Targeted Killer (ATAK) cells diminishes their replicative capacity and markedly increases the efficiency with which they kill Candida albicans, consistent with the cells' maturation towards mature granulocytes. Administration of ATAK cells substantively improves the survival of neutropenic mice infected with C. albicans or Aspergillus fumigatus. Therefore, ATAK cells are capable of recapitulating neutrophil host-defense functions in vitro and in vivo. We hypothesize that ATAK cells are effective at treating disseminated candidiasis because they circulate to infected tissues and there kill fungi by oxidative and/or non-oxidative mechanisms. We also hypothesize that insertion of an inducible suicide trap will enable rapid purging of the cells from a host when desired, thereby enhancing the safety of the strategy. We now seek to define the mechanisms by which ATAK cells kill fungi, to define the ability of an inducible suicide trap to purge the cells in vivo, and to define ATAK cell circulation, persistence, and efficacy in mice infected with a variety of fungal pathogens. The specific aims of this proposal are to: 1) define the mechanisms of ATAK cell anti-fungal activity; 2) transfect ATAK cells with a lentiviral construct containing a suicide trap and a reporter system for tracking cells; 3) define the in vivo circulation, toxicities, and life-span of ATAK cells in neutropenic mice using clinical, laboratory, metabolomic, and histopathological evaluations in two species (mice and rabbit); and 4) define the breadth of protection of ATAK cells against three highly lethal mold infections: aspergillosis, mucormycosis, or fusariosis. Definition of the mechanism of protection and toxicities of ATAK cells will elucidate phagocytic host defense mechanisms against fungi in general, and is critical to future enhancement of ATAK efficacy. Furthermore, insertion of a suicide trap and luminescence marker will enable us to eliminate any residual ATAK cells when desired and to monitor the cells in real- time in vivo. We will also determine the breadth of efficacy of ATAK cells, and define long-term toxicity of the cells in the murine model using clinical, laboratory, metabolomic, and histopathological markers. Ultimately, the studies proposed will provide a foundation for a unique, cutting-edge strategy to recapitulate neutrophil functions in myeloablated hosts with invasive fungal infections.